p53 is an important tumor suppressor that is mutated in more than 50% of human cancers. A major function of p53 is to activate the expression of its target genes, such as p21 and PUMA in response to cellular stress, thereby regulating cell cycle progression and apoptosis. p53 activity is mainly regulated by posttranslational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and methylation. Among them, Mdm2-induced ubiquitination of p53 induces p53 nuclear export and degradation, which is a major cellular mechanism that regulates p53 activity. However, the process of reversing ubiquitination of p53, i.e. deubiquitination, is not well understood. We have found that the ubiquitin-specific protease USP10 deubiquitinates p53 in vitro and in cells. USP10 is required for the stabilization p53 in both unstressed cells and cells subjected to DNA damage. USP10 predominately localizes in the cytoplasm in unstressed cells. In response to DNA damage, USP10 is stabilized and translocates into the nucleus following DNA damage, and ATM-dependent phosphorylated of USP10 is important for USP10 translocation and stabilization. Furthermore, we found that USP10 suppresses tumor cell growth in cells with wild-type p53, while promoting tumor cell growth in cells with mutant p53. Finally, we found loss of USP10 expression in high percentage of renal cell carcinoma cases. Based on these preliminary results, we hypothesize that USP10 is an important regulator of p53, and USP10 suppresses tumorigenesis through p53. Therefore, it is important to further investigate the regulation of the USP10-p53 pathway in cells and in model systems. We propose the following Specific Aims: 1. Study the regulation of USP10-p53 pathway by Ras and G3BP1. G3BP1 (Ras- GTPase-activating protein SH3-domain-binding protein), a downstream effector of Ras, has previously been shown to inhibit USP10 in vitro. We hypothesize that the Ras-G3BP1 pathway regulates p53 stability through USP10, and will test this hypothesis in this aim. 2. Study the regulation of USP10 by the DNA damage response pathway. USP10 predominately localizes in the cytoplasm of unstressed cells. However, following DNA damage, USP10 translocates to the nucleus and becomes stabilized following DNA damage, which contributes to p53 activation. We have shown that ATM-dependent phosphorylation of USP10 is required for USP10 stabilization and translocation, however the mechanisms underlying these events are not entirely clear. We found that USP10 is also sumoylated in a phosphorylation-dependent manner, suggesting that USP10 sumoylation might be important for USP10 stabilization and translocation. We will study the functional significance and regulation of USP10 sumoylation in this aim. 3. Examine the role of USP10 in tumorigenesis. Since USP10 positively regulates p53, we hypothesize that USP10 functions as a tumor suppressor. Consistent with this hypothesis, USP10 can suppress cancer cell growth in p53-dependent manner and USP10 expression is downregulated in high percentage of renal cell carcinoma samples. We will examine the role of USP10 in tumorigenesis in vivo using USP10 knockout mice as a model system. In summary, these studies will reveal novel mechanisms of p53 regulation and tumor suppression. They will also lay the base for future molecular intervention for cancer therapy.